NKG2D, a lectin like immunoreceptor expressed by NK cells and cytotoxic T cells, triggers cytotoxicity and cytokine production by NK cells and enhances T cell responses. Ligands for NKG2D are class I MHC-like molecules including the Rae1/Mult1/ULPB family in humans and mice, and the MICA/MICB ligands in humans. The ligands are poorly expressed by normal cells and induced in stressed cells, cancer cells and pathogen- infected cells. Ligand induction is related to disease-associated signals. In addition to the DNA damage response, which induces ligand expression in transformed cells, we have recently uncovered two additional mechanisms of ligand regulation, which we propose to investigate in this proposal. Two of the aims are directed at developing a deeper molecular understanding of pathways that regulate NKG2D ligands. Aim 1 is to identify the regulatory proteins and E3 ligases that mediate a novel mode of NKG2D ligand regulation, whereby the protein is rapidly degraded in normal cells, but stabilized in stressed cells exposed to heat shock or UV light, or in cancer cells. Aim 2 is to probe a newly discovered mechanism that links cellular proliferation to transcription of the Rae1e NKG2D ligand, and entails identification of the responsible transcription factors and probing instances where NKG2D ligands are repressed in advanced cancer cells. The third aim grew from the discovery that cellular proliferation is apparently a sufficient signal to induce Rae1 expression, along with preliminary data that suggest that such expression is not always sufficient to render cells sensitive to NK elimination in vivo. These findings led to the hypothesis that multiple disease associated signals must converge to render cells sensitive, and as such represent a cancer- associated pattern. Whereas proliferation may induce NKG2D ligands, other signals associated with tumor suppressor activation may be necessary for actual elimination of nascent tumor cells. The third aim proposes a suite of experiments to test this hypothesis in the context of a liver tumor system in which tumor suppressor function can be switched on and off with doxycycline. The proposed experiments will lead to a deeper understanding of how disease-associated signals are integrated to regulate the sensitivity of cells to immune surveillance. The molecular studies should identify new targets for therapeutic augmentation of the immune response in the context of cancer or infections, or for dampening NKG2D-dependent responses in the context of chronic inflammatory diseases.